A-ZIP53, a dominant negative reveals the molecular mechanism of heterodimerization between bZIP53, bZIP10 and bZIP25 involved in Arabidopsis seed maturation

Prateek Jain,Vikas Rishi,Jagdeep Singh,Koushik Shah,Charles Vinson,Nishtha Sharma,Raminder Kaur

doi:10.1038/s41598-017-14167-5

Abstract

In Arabidopsis, maturation phase, an intricate process in seed formation is tightly regulated by the DNA binding activity of protagonist basic leucine zipper 53 (bZIP53) transcription factor and its heterodimerizing partners, bZIP10 and bZIP25. Structural determinants responsible for heterodimerization specificity of bZIP53 are poorly understood. Analysis of amino acid sequences of three bZIPs does not identify interactions that may favor heterodimerization. Here, we describe a designed dominant negative termed A-ZIP53 that has a glutamic acid-rich amphipathic peptide sequence attached to N-terminal of bZIP53 leucine zipper. Circular dichroism (CD) and mass spectrometry studies with equimolar mixture of three bZIP proteins in pairs showed no heterodimer formation whereas A-ZIP53 interacted and formed stable heterodimers with bZIP53, bZIP10, and bZIP25. A-ZIP53 electrostatically mimics DNA and can overcome repulsion between basic DNA binding regions of three bZIP proteins. Gel shift experiments showed that A-ZIP53 can inhibit the DNA binding of three proteins. CD studies demonstrated the specificity of A-ZIP53 as it did not interact with bZIP39 and bZIP72. Transient co-transfections in Arabidopsis protoplasts showed that A-ZIP53 inhibited three bZIPs and their putative heterodimers-mediated transactivation of GUS reporter gene. Furthermore, four newly designed acidic extensions were evaluated for their ability to interact with three bZIPs.

Highlights

BZIP transcription factors (TFs) are eukaryote specific proteins that bind to short but specific DNA sequences as a dimeric parallel coiled coil and regulate gene expression
Coiled coil nomenclature with each heptad represented by gabcdef is shown at the top of the leucine zipper dimerization domain. bZIP25 and bZIP10 are predicted to be 8 heptads long with their C-terminus boundaries defined by the absence of proline. basic leucine zipper 53 (bZIP53), due to the absence of a proline and two consecutive glycines, presence of charged amino acids in g and e position, and hydrophobic amino acids in a and d positions is predicted to be more than 8 heptads long
67 bZIPs of Arabidopsis thaliana were placed in 20 families (A-T) based on their predicted dimerizing properties10. bZIP25 and bZIP10 were included in G family whereas bZIP53 was placed in H family

Summary

Introduction

BZIP transcription factors (TFs) are eukaryote specific proteins that bind to short but specific DNA sequences as a dimeric parallel coiled coil and regulate gene expression. BZIP TFs have transactivation domain present either at N- or C-terminal[1]. These TFs homo- or heterodimerize via a monomer or dimer pathway and occupy major grooves of DNA4–7. A-ZIP53 contains a rationally designed glutamic acid-rich peptide extension that replaces the N-terminal basic DNA binding domain of bZIP53. Since A-ZIP53 and its four derivate can heterodimerize with bZIP53, bZIP10 and bZIP25 and inhibit their DNA binding, these proteins may be used to study dimerization specificity of these bZIPs in vitro and in vivo

Methods

Results

Conclusion